The overall goal of this SBIR AT application is to develop a multi-adjuvant liposomal vaccine formulation that targets dendritic cells and can be applied to our current vaccine programs. The innovation is a dual adjuvant system where VesiVax(R) lipid vesicles are formulated with a TLR9 agonist (CpG) and CD40 Ligand fused to the Company's proprietary Hydrophobic Domain (HD) technology. The formulations, including testing of single adjuvants, will be assessed with influenza and Herpes Simplex 2 viral antigens in vivo in proven mouse models. We have shown that the TLR4 agonist, MPL has an immunostimulatory effect and hypothesize that other TLR agonists and receptor-binding ligands will influence the immune response elicited by target antigens. The ultimate goal of this program is to build an immunogenic liposome formulation that is broadly applicable to antigens, regardless of whether a predominantly Th1 or Th2 type of immune response is desired. The optimal formulation will be utilized to advance an influenza or Herpes Simplex Virus 2 (HSV2) vaccine candidate towards the clinic. Vaccines based on the VesiVax(R) system can be engineered using two formats: 1) a target antigen or adjuvant protein can be engineered to be expressed as a fusion protein with the HD which facilitates incorporation directly into the liposome or; 2) a target antigen or adjuvant in the form of a peptide or carbohydrate that can be attached via conjugation to the surface of the liposomes, or conjugatable adjuvant lipid vesicles (CALVs). The Company has assembled an internal team, as well as an external collaborator, with expertise in key core competencies in recombinant protein production, understanding structural and functional interactions of proteins and peptides with lipid bilayers in order to prepare liposomes with the appropriate properties, and to analyze the liposomes and lipid, protein, and adjuvant components to confirm the composition. Our collaborator for the proposed in vivo studies has been testing VesiVax(R)-TLR4 vaccines in established challenge models for influenza and HSV2. The approach to evaluating the proposed dual adjuvant system involves creating the CD40L and TLR9 adjuvanted formulations and testing their effect formulated in vaccines containing the influenza M2e or HSV2 gD antigens. Using methods already developed by the Company for M2e-HD and gD-HD, we will prepare CD40L as an HD fusion expressed in E. coli, and we will prepare the selected CpG oligonucleotide sequence either synthesized coupled to a lipid or with a free thiol or free amine on the end to attach to lipid. We will use ou published female mouse model of intravaginal HSV2 infection and mouse influenza intranasal challenge model. These models will allow us to test each adjuvant in separate formulations as well as combined. Ultimately, we hypothesize that the combination of the two adjuvants will stimulate stronger protective immune responses, thus leading to an optimized commercial formulation for our vaccine programs. The first Milestone in this proposal is the production of liposomes formulated with CpG, CD40L-HD, and both CpG and CD40L-HD. The second Milestone is the choice of an optimal formulation by testing in the two infectious disease models. In Phase 2, we will develop commercial production and analytical methods and conduct nonclinical Pharmacology/Toxicology studies to support the filing of an IND. A secondary application of the new adjuvant system would be to make it available to vaccinologists and immunologists in a kit format. A Foresight Science & Technology market research report entitled Liposomal Adjuvant for Vaccine Research was commissioned by the Company. The report identified lipid-based adjuvants as having a very broad range of potential applications and corresponding to more than 80% of the market for vaccines in development, which is estimated to reach more than $18 billion by 2015. Using this statistic, it is estimated that the total addressable market for lipid-based adjuvants may be over $14.4 billion by 2015. PUBLIC HEALTH RELEVANCE: With the continued emergence of new pathogens, the prevalence of certain viral infections, and the threat of bioterrorism, it is imperative to continu to optimize vaccine performance. Molecular Express is developing vaccines to two viral targets, influenza A virus (IAV) and Herpes Simplex Virus 2 (HSV2). Herpes genitalis caused by HSV2 is one of the most common, sexually transmitted diseases in humans, with as many as 1 out of 6 Americans having been infected. The disease affects both normal and immunosuppressed adults, and is associated with increased susceptibility to the human immunodeficiency virus, serious clinical disease in neonates following transmission of virus from their infected mothers, and increased likelihood of developing cervical cancer versus non-infected women. IAV infections have caused the deaths of millions of humans worldwide. While generally effective against individual viral subtypes, vaccines that protect against infection by IAV are difficult to manufacture rapidly and demonstrate limited activity against different subtypes due to antigenic shift and drift in the surface antigens of the virus. In addition, the recent emergence of highly virulent strains of IAV could result in a devastating human pandemic before effective vaccine candidates could be developed. Vaccines that provide broad protection against infection with emerging strains of IAV and are manufactured in a manner that allows the vaccine to be rapidly and effectively updated in response to new strains of IAV would be a major advance. Clearly there are critical needs to develop effective vaccines against these two diseases. There is unexplored potential to optimize our current influenza A virus (IAV) and Herpes Simplex Virus 2 (HSV2) vaccine candidates by targeting immunostimulation through Dendritic Cell (DC) activating pathways. We will track the immunostimulatory properties of vaccine formulations using established in vivo viral challenge models. We hypothesize that by combining a Toll-like receptor 9 agonist, CpG, with CD40 Ligand, DC cells will facilitate an augmented response to VesiVax(R) liposomal vaccines. Testing of the DC-targeted dual adjuvant approach will be conducted on two vaccines, IAV Matrix 2 protein ectodomain (L-M2e1-15-HD) and HSV2 surface glycoprotein (L-gD1-306), which generates predominantly Th2 and predominantly Th1 responses, respectively. The goal of this project is to understand and optimize vaccine formulations that are broadly applicable to our vaccine programs. Thus, successful execution of the studies proposed in this application could lead to new and/or improved vaccines for treatment of a variety of infectious diseases or cancers.